Compositions comprising ornithine and phenylacetate or phenylbutyrate for treating hepatic encephalopathy

ABSTRACT

The present invention relates to use of ornithine in the manufacture of a medicament for use in combination with at least one of phenylacetate and phenylbutyrate for preventing or treating liver decompensation or hepatic encephalopathy. The invention also relates to use of at least one of phenylacetate and phenylbutyrate in the manufacture of a medicament for use in combination with ornithine for preventing or treating liver decompensation or hepatic encephalopathy.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/720,268,which was the National Stage of International Application No.PCT/GB2005/004539, filed Nov. 28, 2005, which claims the benefit ofUnited Kingdom Patent Application Nos. 0426141.8 and 0426142.6, bothfiled on Nov. 26, 2004.

FIELD OF THE INVENTION

The present invention relates to the prevention or treatment of liverdecompensation or hepatic encephalopathy.

BACKGROUND OF THE INVENTION

Chronic liver disease is characterised by the gradual destruction ofliver tissue over time, whereby healthy and regenerating liver tissue isslowly replaced with scar and necrotic tissue. This is known as livercirrhosis. Normal liver function is impaired and the scar tissueprogressively diminishes blood flow through the liver. As normalregenerating liver tissue is lost, nutrients, hormones, drugs and toxinsare no longer effectively processed.

This can result in symptoms including abnormal clearance of proteinsabsorbed through the intestinal tract, leading to accumulation ofammonia; abnormal excretion, leading to an accumulation of bilirubin inthe blood, producing jaundice; increased sinusoidal pressure, leading tofluid accumulation in the abdomen (ascites); and portal hypertension(and portosystemic shunting) wherein scarred liver tissue acts as abarrier to blood flow, leading to increased portal blood pressure andoesophageal varices.

Patients with chronic liver disease can be in a fairly stable clinicalstate and exhibit few or no symptoms. However, such patients are at riskof an abrupt deterioration in their condition which can lead toacute-on-chronic liver failure. This transition from a “compensated”state, where the liver is able to function, albeit at a reduced level,to a “decompensated” state, where liver function fails, involves theeffect of precipitating events. Precipitating events associated withchronic liver disease include gastrointestinal bleeding, infection(sepsis), portal vein thrombosis and dehydration.

For example, 50% of patients with cirrhosis of the liver haveoesophageal varices and in a third of these patients, the oesophagealvarices will burst and cause gastrointestinal bleeding within two yearsof diagnosis (Grace N D (1992) Gastroenterol Clin North Am 21: 149-161).An upper gastrointestinal bleed is known to increase the susceptibilityto life-threatening complications such as bacterial peritonitis, sepsis,renal failure and hepatic encephalopathy (Tenn et al. (1997)Gastroenterology 112: 473-482; Garden et al. (1985) Br J Surg 72: 91-95;Pauwels et al. (1996) Hepatology 24: 802-806; Bleichner et al. (1986) BrJ Surg 73: 724-726) resulting in the death of about 30% of patientsdespite adequate control of bleeding (Grace 1992 supra).

Hepatic encephalopathy (HE) is a complex neuropsychiatric disorder thatoccurs in diverse clinical situations such as acute or chronic liverdisease and spontaneous portosystemic venous shunting. In the earlystages of hepatic encephalopathy subtle mental changes occur such aspoor concentration, confusion and disorientation. In severe cases,hepatic encephalopathy can lead to stupor, coma, brain swelling(cerebral edema) and death. In the case of patients who develop HE as aresult of chronic liver disease, the onset of HE is often the result ofa clinically precipitating event such as gastrointestinal bleeding,sepsis (infection), portal vein thrombosis or dehydration.

Gastrointestinal bleeding and portosystemic shunting allows toxicsubstances, which are usually metabolised by the liver, to bypass theliver, enter the systemic circulation and cross the blood-brain barrierto exert direct or indirect neurotoxic effects on the central nervoussystem. Ammonia accumulation is thought to play an important role in theprogression of hepatic encephalopathy and multiorgan failure(respiratory failure, cardiovascular failure, kidney failure). Inaddition to ammonia, septicaemia (or bacterial peritonitis) whichdevelops soon after a gastrointestinal bleed is also likely to be acontributing factor to hepatic encephalopathy.

Liver decompensation can then lead to multiorgan failure and hepaticencephalopathy. In the early stages of hepatic encephalopathy subtlemental changes such as poor concentration or the inability to constructsimple objects occurs. In severe cases, hepatic encephalopathy can leadto stupor, coma, brain swelling and death.

The prognosis for patients with chronic liver disease is difficult toestimate because the condition has many causes. Preventative measures tominimise progression from the compensated state to the decompensatedstate include avoidance of further causative agents which will worsenthe condition, such as complete abstinence from alcohol and vaccinationagainst hepatitis A and B.

However, once liver decompensation occurs, the chances of survival arereduced and liver transplantation is the only treatment that can extendlife. Since it is liver decompensation that leads to a reduced lifeexpectancy, it is highly desirable to prevent liver decompensation fromoccurring.

A common therapy for patients with hepatic encephalopathy involvesstrategies to reduce the concentration of ammonia. These includerestriction of dietary protein intake; administration of lactulose,neomycin, L-ornithine L-aspartate (LOLA), or sodium benzoate; andcleansing enemas.

SUMMARY OF THE INVENTION

The present invention concerns the use of ornithine and at least one ofphenylacetate and phenylbutyrate to prevent or treat liverdecompensation or hepatic encephalopathy (HE) in patients. Isoleucinemay also be administered to those patients further having an isoleucinedeficiency attributable, for example to gastrointestinal bleeding.Accordingly, the invention provides:

use of omithine in the manufacture of a medicament for use incombination with at least one of phenylacetate and phenylbutyrate forpreventing or treating liver decompensation or hepatic encephalopathy;

use of at least one of phenylacetate and phenylbutyrate in themanufacture of a medicament for use in combination with ornithine forpreventing or treating liver decompensation or hepatic encephalopathy;

use of omithine and at least one of phenylacetate and phenylbutyrate inthe manufacture of a medicament for preventing or treating liverdecompensation or hepatic encephalopathy;

products containing ornithine and at least one of phenylacetate andphenylbutyrate for simultaneous, separate or sequential use forpreventing or treating liver decompensation or hepatic encephalopathy;

a pharmaceutical composition comprising omithine and at least one ofphenylacetate and phenylbutyrate;

an agent for preventing or treating liver decompensation or hepaticencephalopathy, comprising ornithine and at least one of phenylacetateand phenylbutyrate; and

a method of treating a patient having or at risk of having liverdecompensation or hepatic encephalopathy, which method comprisesadministering an effective amount of ornithine and at least one ofphenylacetate and phenylbutyrate to said patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that neutrophil function is altered in patients withcirrhosis and worsens with increasing severity of liver disease.

FIG. 2 shows that ammonia reduces neutrophil phagocytosis.

FIG. 3 shows that ammonia reduces neutrophil chemotaxis.

FIG. 4 shows that the effect of ammonia on neutrophil phagocytosis canbe reversed by interventions.

FIG. 5 shows that a simulated gastrointestinal bleed reduces neutrophilchemotaxis which can be partially reversed by administration ofisoleucine.

FIG. 6 shows that a simulated bleed reduces protein synthesis andstimulates isoleucine oxidation inappropriately.

FIG. 7 shows that administration of isoleucine during a simulated bleedenhances protein synthesis but does not reduce ammonia concentration.

FIG. 8 shows that administration with LOLA reduces ammonia concentrationbut allows ammonia to regenerate.

FIG. 9 shows that active removal of glutamine prevents the secondaryrise in ammonia concentration.

FIG. 10 shows that phenylacetate binds glutamine to make an excretablecompound and prevents the secondary rise in ammonia.

FIG. 11 shows the effect of ornithine and phenylbutyrate on ammonialevels in patients with advanced cirrhosis.

FIG. 12 shows the effect of ornithine and phenylbutyrate on glutaminelevels in patients with advanced cirrhosis.

FIG. 13 shows the changes in mental state of patients treated withplacebo, O, P or O+P.

FIG. 14 shows the effect of ornithine, phenylbutyrate and isoleucine onammonia levels in patients with advanced cirrhosis.

FIG. 15 shows the effect of ornithine, phenylbutyrate and isoleucine onglutamine levels in patients with advanced cirrhosis.

FIG. 16 shows the effect of ornithine, phenylbutyrate and isoleucine onglycine levels in patients with advanced cirrhosis.

FIG. 17 shows the effect of ornithine, phenylbutyrate and isoleucine onisoleucine levels in patients with advanced cirrhosis.

FIG. 18 shows the effect of ornithine, phenylbutyrate and isoleucine onornithine levels in patients with advanced cirrhosis.

FIG. 19 shows the effect of ornithine and phenylbutyrate on arterialammonia in the bile duct ligated rat model.

FIG. 20 shows the effect of omithine and phenylbutyrate on plasmaornithine in the bile duct ligated rat model.

FIG. 21 shows the effect of omithine, phenylbutyrate and isoleucine onarterial plasma ammonia levels in a hyperammonaemic acute liver failurerat model.

FIG. 22 shows muted arterial ammonia increase in the devascularized pigmodel of acute liver failure with OP treatment.

FIG. 23 shows that ammonia is being taken from the blood by the musclein the O and the OP treated animals (samples were taken from the femoralvein-artery). In contrast, the placebo and the P alone animals shows anincrease in ammonia production by the muscle.

FIG. 24 shows that ammonia is produced by the gut in all animals exceptthe OP treated animal (samples were taken from the portal drainedviscera-artery).

FIG. 25 shows that muscle glutamine release is increased by O but not Pused in isolation. OP caused a markedly greater release of muscleglutamine (thereby trapping ammonia as glutamine in the muscle).

FIG. 26 shows that gut glutamine uptake is enhanced by O, but reduced byOP (thereby reduced generation of ammonia in the gut).

FIG. 27 shows that arterial omithine levels increase in the two animals(O alone and OP groups) to which it is administered.

FIG. 28 shows that arterial glutamine levels rise with O, but less sowith OP.

FIG. 29 shows that the combination of OP prevents the increase in theammoniagenic amino acid glycine.

FIG. 30 shows that ornithine alone caused an increase in brain water,phenyl acetate induced a small reduction in brain water, while incombination these agents bring about a substantial reduction in brainwater (% control).

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

The present invention is concerned with the early treatment of patientswith liver disease, before development of liver decompensation and thusbefore hepatic encephalopathy has occurred, to prevent or delay theonset of liver decompensation. Alternatively, the present invention isconcerned with treatment of hepatic encephalopathy by effectivelyreducing ammonia concentration and maintaining neutrophil function.

Subjects to be Treated

The present invention is concerned with the prevention or treatment ofliver decompensation or hepatic encephalopathy. The subject's liver maytherefore be in the compensated state. The subject may have chronicliver disease. The subject may have liver cirrhosis. The subject mayhave acute liver failure. The subject to be treated may have hepaticencephalopathy.

The onset of both acute and chronic liver disease may be due to axenobiotic cause. For example, the subject may have been exposed to achemical, drug or some other agent which causes liver damage. Thesubject may have a reaction to an over-the-counter, prescriptive or“recreational” drug which causes liver damage. The subject may have beentaking Rezulin™ (troglitazone; Parke-Davis), Serzone™ (nefazodone;Bristol-Myers Squibb) or other drugs thought to cause liver damage. Thesubject may be one who has had an overdose of a particular drug orexceeded the recommended dosage of a drug capable of causing liverdamage. For example, the subject may have taken an overdose ofparacetamol. The subject may have been exposed to chemicals which cancause liver damage such as, for example, at their place of work. Forexample, the subject may have been exposed to such chemicals in anindustrial or agricultural context. The subject may have consumed plantswhich contain compounds which can cause liver damage, in particular thismay be the case where the subject is an animal, such as a herbivore. Forexample, the subject may have consumed a plant containing pyrrolizidinealkaloid such as ragwort. The subject may have been exposed toenvironmental toxins thought to cause liver disease.

Drug-related liver toxicity comprises more than 50% of all cases withacute liver disease (acute liver failure). Acetaminophen-(also known asparacetamol and N-acetyl-p-aminophenol) toxicity is the most commoncause of acute liver failure in the United States and Great Britain.Long-term moderate to heavy alcohol users who take acetaminophen intherapeutic or modestly excessive doses are at risk of severe hepaticinjury and possibly acute liver failure. Alcohol use potentiates thetoxic effects of acetaminophen. Idiosyncratic drug toxicity alsocontributes to acute liver failure. Idiosyncratic drug toxicity isthought to be a hypersensitivity response wherein the subject respondsto a drug in a pharmacologically abnormal way. This abnormal responsecan lead to acute liver failure.

The acute liver failure or chronic liver disease may be caused byinfection with a pathogenic organism. For example, the liver disease maybe due to viral infection. In particular, the subject may be infected,or have been infected, with a virus which causes hepatitis. The subjectmay have chronic viral hepatitis. The virus may, for example, behepatitis B, C or D virus. In some cases, and in particular where thesubject has viral hepatitis, the subject may also be infected with HIV-Ior II. The subject may have AIDS. It is possible that the subject mayhave been, or be, infected with other organisms which cause liverdisease and in particular those which are present in the liver duringsome stage of their life cycle. For example, the subject may have, orhave had, liver fluke.

The subject may have an inherited disease which causes, or increases therisk of, chronic liver disease. For example, the subject may have one ormore of hepatic hemochromatosis, Wilson's disease or α-1-antitrypsindeficiency. The subject may have an inherited disorder which causes somekind of structural or functional abnormality in the liver whichincreases the likelihood of liver fibrosis. The subject may begenetically predisposed to develop an autoimmune disorder which damagesthe liver and hence which can contribute to liver fibrosis.

The chronic liver disease may be alcohol-induced. A man or woman to betreated may be, or have been, an alcoholic. He or she may be, or havebeen, consuming on average 50 or more units of alcohol per week, 60 ormore units of alcohol per week, 75 or more units of alcohol per week andeven 100 or more units of alcohol per week. The man or woman may be, orhave been, consuming on average up to 100 units of alcohol per week, upto 150 units of alcohol per week and even up to 200 units of alcohol perweek. The measurement of one unit of alcohol differs from country tocountry. Here, one unit equals 8 grams of ethanol in accordance with theUnited Kingdom standard.

The man or woman may have been consuming such levels of alcohol for 5 ormore years, 10 or more years, 15 or more years or 20 or more years. Thesubject may have been consuming such levels of alcohol for up to 10years, up to 20 years, up to 30 years and even up to 40 years. In casesof alcohol-induced liver cirrhosis the subject may be aged, for example,25 years or over, 35 years or over, 45 years or over and even over 60years.

The subject may be male or female. Women may be more susceptible to theadverse effects of alcohol than men. Women can develop alcoholic chronicliver disease in a shorter time frame and from smaller amounts ofalcohol than men. There seems to be no single factor to account forincreased susceptibility to alcoholic liver damage in females, but theeffect of hormones on the metabolism of alcohol may play an importantrole.

In other embodiments of the invention, the subject may have one or moreof a number of other conditions known to result in liver damage such as,for example, primary biliary cirrhosis, autoimmune chronic activehepatitis, and/or schistosomiasis (parasitic infection). The subject mayhave or have had a bile duct blockage. In some cases, the underlyingcause of chronic liver disease may not be known. For example the subjectmay have been diagnosed as having cryptogenic cirrhosis. In oneembodiment, the subject may be suspected of having any of the conditionslisted herein.

Methods for diagnosing chronic liver disease, acute liver failure andhepatic encephalopathy are well known in the art and in particular toclinicians and veterinarians in the field. Preferably, the subject willhave been diagnosed as having a liver disease and hepaticencephalopathy, for example by a medical or veterinarian professional.The subject may display one or more symptoms associated with liverdisease such as one or more of jaundice, ascites, skin changes, fluidretention, nail changes, easy bruising, nose bleeds, oesophagealvarices, and in male subjects may have enlargement of breasts. Thesubject may display exhaustion, fatigue, loss of appetite, nausea,weakness and/or weight loss. The subject may also display one or moresymptoms associated with hepatic encephalopathy such as one or more ofconfusion, disorientation, dementia, stupor, coma, cerebral edema,multiorgan failure (respiratory failure, cardiovascular failure orkidney failure), muscle stiffness/rigidity, seizures or speechimpairment. The subject to be treated may or may not be taking otherdrugs to treat liver disease. The subject to be treated may be at riskof developing hepatic encephalopathy.

The liver disease may have been, or be, confirmed by physicalexamination including techniques such as ultrasound. Liver biopsies mayhave been taken to look for build up of fibrosis, necrotic cells,cellular degeneration and/or inflammation and other characteristicfeatures of liver disease. Liver function may have been assessed in thesubject to determine whether this is compromised in the subject. Thenature and underlying cause of the liver disease may be characterized.Any history of exposure to causative agents of liver disease may bedetermined.

The subject to be treated may be at risk for hepatic encephalopathicepisodes, for example patients who are awaiting liver transplants,surgical and/or portal hypertension patients. A person at risk forhepatic encephalopathic episodes is a person who has not suffered anyhepatic encephalopathic episodes or has not suffered any hepaticencephalopathic episode for an extended period of time (about 12 weeksor longer), but has a disorder or medical condition which creates a riskof hepatic encephalopathic episodes. A hepatic encephalopathic episodeis a clinical condition characterised by the presence of cerebraldysfunction in patients with liver disease or dysfunction. There is awide spectrum of mental disturbances in hepatic encephalopathy whichrange from minimal where the main effects are a reduction in the qualityof life, to overt which leads to coma and ultimately death.

Scoring systems may be used to assess the severity of liver disease andhepatic encephalopathy and also the prognosis of subjects. TheChild-Pugh, West Haven Criteria, Glasgow Coma Scale or modifiedChild-Pugh scoring system may be used. Alternatively, the (APACHE) IIscoring system may be used. Points are assigned to parameters includingserum bilirubin levels, serum albumin levels and to signs includingpresence of ascites or encephalopathy. Subjects to be treated may beclassified in Child-Pugh class A, B or C. Generally subjects to betreated are classified in Child-Pugh class C.

A man or woman to be treated may be aged, for example from 25 to 80years. In one embodiment, the man or woman is aged from 45 to 70 years.In another embodiment, the man or woman is aged from 25 to 44 years. Ina further embodiment, the man or woman is aged over 65 years.

The invention does have veterinary use, however. The subject to betreated may be a farm animal for example, a cow or bull, sheep, pig, ox,goat or horse or may be a domestic animal such as a dog or cat. Thesubject may or may not be an animal model for liver disease. The animalmay be any age, but will often be a mature adult subject.

Formulation

The amino acids used in the present invention may be pure crystallineamino acids. In general, the amino acids are in the L-form, rather thanthe D-form, or a mixture of D and L. Isolated forms of the amino acidsare typically used. Any active form of the amino acid may be used toprevent or treat the liver decompensation or hepatic encephalopathy. Apharmaceutically acceptable form of the amino acid may be used. Theamino acids may be employed as free amino acids or amino acid salts orderivatives.

Ornithine may be in pure crystalline amino acid form. In general,omithine is in the L-form, rather than the D-form, or a mixture of D andL. Isolated forms of ornithine are typically used. Any active form ofornithine may be used or a pharmaceutically acceptable form of ornithinemay be used. Ornithine may be employed as a free amino acid or an aminoacid salt or derivative.

Typically, ornithine is used as a single, monomeric amino acid.Ornithine may be used in salt form, for example ornithine hydrochloridemay be used. Ornithine may be in the form of a physiologicallyacceptable salt in free form. Therefore, the ornithine or the omithinesalt are typically not chemically bound, or covalently linked to anyother agent.

Derivatives of ornithine may be used. For example, keto or hydroxyanalogs of ornithine may be administered as sodium or calcium salts.Keto acids of omithine include ornithine ketoglutarate, ornithineketoleucine and omithine ketovaline. Salts or derivatives of ornithinemay be used in place of or in addition to free omithine.

At least one of phenylacetate and phenylbutyrate may be used.Phenylacetate and/or phenylbutyrate may be in physiologically acceptablesalt form, such as an alkali metal or alkaline earth metal salt. Thesalt may be sodium phenylacetate or sodium phenylbutyrate. The salt formof phenylacetate and phenylbutyrate may be in free form. Therefore thephenylacetate and phenylbutyrate or phenylacetate salt andphenylbutyrate salt are typically not chemically bound, or covalentlylinked to any other agent.

Optionally isoleucine is used. Isoleucine may be in pure crystallineamino acid form. In general, isoleucine is in the L-form, rather thanthe D-form, or a mixture of D and L. Isolated forms of isoleucine aretypically used. Any active form of isoleucine may be used or apharmaceutically acceptable form of isoleucine may be used. Isoleucinemay be employed as a free amino acid or an amino acid salt orderivative.

Typically, isoleucine is used as a single, monomeric amino acid.Isoleucine may be used in salt form, for example isoleucinehydrochloride may be used. Isoleucine may be in the form of aphysiologically acceptable salt in free form. Therefore, the isoleucineor the isoleucine salt are typically not chemically bound, or covalentlylinked to any other agent.

Pharmaceutical Compositions

The ornithine and the phenylacetate and/or phenylbutyrate are typicallyformulated for administration with a pharmaceutically acceptable carrieror diluent. The ornithine and the phenylacetate and/or phenylbutyratemay thus be formulated as a medicament with a standard pharmaceuticallyacceptable carrier(s) and/or excipient(s) as is routine in thepharmaceutical art. The exact nature of the formulation will depend uponseveral factors including the desired route of administration.Typically, ornithine and the phenylacetate and/or phenylbutyrate areformulated for oral, intravenous, intragastric, intravascular orintraperitoneal administration.

The pharmaceutical carrier or diluent may be, for example, an isotonicsolution such as physiological saline. Solid oral forms may contain,together with the active compound, diluents, e.g. lactose, dextrose,saccharose, cellulose, corn starch or potato starch; lubricants, e.g.silica, talc, stearic acid, magnesium or calcium stearate, and/orpolyethylene glycols; binding agents; e.g. starches, gum arabic,gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginatesor sodium starch glycolate; effervescing mixtures; dyestuffs;sweeteners; wetting agents, such as lecithin, polysorbates,laurylsulphates; and, in general, non-toxic and pharmacologicallyinactive substances used in pharmaceutical formulations. Suchpharmaceutical preparations may be manufactured in known manner, forexample, by means of mixing, granulating, tabletting, sugar-coating, orfilm-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions orsuspensions. The syrups may contain as carriers, for example, saccharoseor saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a naturalgum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol. The suspensions orsolutions for intramuscular injections may contain, together withornithine and at least one of phenylacetate and phenylbutyrate, apharmaceutically acceptable carrier, e.g. sterile water, olive oil,ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitableamount of lidocaine hydrochloride.

Medicaments of the invention can comprise omithine as the only aminoacid component. Medicaments of the invention can comprise ornithine andisoleucine as the only amino acid components. The medicament may consistessentially of omithine and at least one of phenylacetate andphenylbutyrate. The medicament may consist essentially of ornithine,isoleucine and at least one of phenylacetate and phenylbutyrate.

The medicament may consist essentially of ornithine, phenylacetateand/or phenylbutyrate and a pharmaceutically acceptable carrier. Such amedicament therefore contains substantially no other amino acid inaddition to ornithine. The medicament may consist essentially ofomithine, isoleucine, phenylacetate and/or phenylbutyrate and apharmaceutically acceptable carrier. Such a medicament thereforecontains substantially no other amino acid in addition to ornithine andisoleucine.

The phenylacetate may be present in an amount from 5 to 100%, forexample from 10 to 50%, or 20 to 40%, by weight of the weight ofomithine. The phenylbutyrate may be present in an amount from 5 to 100%,for example from 10 to 50%, or 20 to 40%, by weight of the weight ofornithine.

However, the medicament may comprise free aspartate, glutamate orarginine in non-peptide form, typically in an insubstantial amount.Generally, the amount by weight of aspartate, glutamate or arginine doesnot exceed the amount by weight of omithine. By an insubstantial amount,it is meant that the amount by weight of aspartate, glutamate orarginine, or a combination of these amino acids, does not exceed 20% byweight of ornithine. Therefore, the medicament may comprisesubstantially no aspartate. In one embodiment, the composition does notcomprise aspartate, glutamate or arginine. Trace amounts of aspartate,glutamate or arginine may be present in the composition. By traceamount, it is meant that the amount by weight of aspartate, glutamate orarginine, or a combination of these amino acids, does not exceed 1% byweight of ornithine. Preferably, the amount by weight of aspartate,glutamate or arginine does not exceed 0.5% by weight of omithine.

In another embodiment, the composition may comprise yet other aminoacids in non-peptide form, typically as the free amino acid or aphysiologically acceptable salt thereof in free form. The amount ofthese other amino acids generally does not exceed the amount by weightof ornithine. For example, the other amino acids may be present in anamount by weight up to 20%, for example from 5 to 20%, of the weight ofomithine. Such other amino acids that may be present in the compositioninclude essential and non-essential amino acids. The composition maycomprise other branched chain amino acids (BCAAs). BCAAs includeisoleucine, valine and leucine. Thus, a composition of the invention mayfurther comprise isoleucine and/or valine and/or leucine.

Treatment

Ornithine and at least one of phenylacetate and phenylbutyrate areadministered in combination to a subject for preventing or delaying theonset of liver decompensation or hepatic encephalopathy. Ornithine andat least one of phenylacetate and phenylbutyrate can thus beadministered in combination to improve the condition of a subject, forexample a subject suffering from chronic liver disease following aprecipitating event. Ornithine and at least one of phenylacetate andphenylbutyrate may be administered in combination to alleviate thesymptoms of a subject, for example the symptoms associated with chronicliver disease in a subject following a precipitating event. Ornithineand at least one of phenylacetate and phenylbutyrate may be administeredin combination to combat or delay the onset of liver decompensation orhepatic encephalopathy.

Ornithine and at least one of phenylacetate and phenylbutyrate may beadministered in combination to a subject for treatment of hepaticencephalopathy. Ornithine and at least one of phenylacetate andphenylbutyrate may be administered in combination to improve thecondition of a patient suffering from hepatic encephalopathy. Ornithineand at least one of phenylacetate and phenylbutyrate may be administeredin combination to alleviate the symptoms associated with hepaticencephalopathy. Ornithine and at least one of phenylacetate andphenylbutyrate may be administered in combination to combat hepaticencephalopathy. Ornithine and at least one of phenylacetate andphenylbutyrate may be administered in combination to prevent an initialhepatic encephalopathic episode in a person at risk of for hepaticencephalopathic episodes. Ornithine and at least one of phenylacetateand phenylbutyrate may be administered in combination lessen theseverity of an initial hepatic encephalopathic episode in a person atrisk of for hepatic encephalopathic episodes. Ornithine and at least oneof phenylacetate and phenylbutyrate may be administered in combinationto delay an initial hepatic encephalopathic episode in a person at riskof for hepatic encephalopathic episodes.

Development of liver decompensation and hepatic encephalopathy involves“precipitating events” (or “acute attacks”). Such precipitating eventsinclude gastrointestinal bleeding, infection (sepsis), portal veinthrombosis and dehydration. The onset of such an acute attack is likelyto lead to hospitalisation. The patient may suffer one of these acuteattacks or a combination of these acute attacks.

A subject who has had or is suspected of having had an acute attack istreated according to the invention with ornithine and phenylacetateand/or phenylbutyrate in combination to prevent progression of the liverto the decompensated state. The invention can therefore prevent themedical consequences of liver decompensation such as hepaticencephalopathy. The ornithine and phenylacetate and/or phenylbutyratemay be used to preserve liver function. Use of ornithine andphenylacetate and/or phenylbutyrate may thus extend the life of apatient with liver disease. In one embodiment, the metabolicconsequences of a gastrointestinal bleed such as hyperammonemia,hypoisoleucemia and reduced protein synthesis in the post-bleedingperiod are prevented.

Typically, treatment of subjects may begin as soon as possible after theonset or the suspected onset of a precipitating event (acute attack).Preferably, treatment of the subject begins prior to repeated acuteattacks. More preferably, treatment of the subject begins following thefirst acute attack.

Treatment is typically given promptly after the start of an acuteattack. Treatment may begin after the symptom(s) of an acute attack orsuspected acute attack have been detected e.g. by a medic such as aphysician, a paramedic or a nurse. Treatment may begin uponhospitalisation of the subject. Treatment may thus begin within 6 hours,within 3 hours, within 2 hours or within 1 hour after the symptom(s) ofan acute attack or suspected acute attack have been detected. Treatmentof the subject may therefore begin from 1 to 48 hours, for example from1 to 36 hours or from 1 to 24 hours after the symptom(s) of an acuteattack or suspected acute attack have been detected.

Treatment may occur for up to 8 weeks, for example up to 6 weeks, up to4 weeks or up to 2 weeks after the symptom(s) of an acute attack orsuspected acute attack have been detected. Treatment may therefore occurfor up to 48 hours, for example for up to 36 hours or for up to 24 hoursafter the symptom(s) of an acute attack or suspected acute attack havebeen detected. Typically, treatment occurs to the time when recoveryfrom the acute precipitating event is evident.

The subject is treated with the ornithine and the phenylacetate and/orphenylbutyrate. Ornithine and at least one of phenylacetate andphenylbutyrate may be administered in combination in a singlemedicament, or separately in two or three different medicaments. Whereornithine and at least one of phenylacetate and phenylbutyrate are to beadministered in a combined medicament, the combination may be preparedimmediately before administration, or may be stored as a combinedmedicament.

Where the ornithine and the phenylacetate and/or phenylbutyrate are tobe administered separately, the medicaments may be administeredsimultaneously or sequentially over a period of time. Two or threeseparate medicaments may be administered over a period of time.

Where two medicaments are administered, ornithine may be administeredfirst, followed by administration of the phenylacetate andphenylbutyrate, the phenylacetate or the phenylbutyrate. Alternatively,the phenylacetate and phenylbutyrate, the phenylacetate or thephenylbutyrate may be administered first, followed by ornithine. Inanother embodiment, a combination of ornithine and phenylacetate may beadministered first, followed by administration of phenylbutyrate.Alternatively, a combination of ornithine and phenylbutyrate may beadministered first, followed by administration of phenylacetate. Inanother embodiment, phenylacetate may be administered first, followed byadministration of a combination of ornithine and phenylbutyrate.Alternatively, phenylbutyrate may be administered first, followed byadministration of a combination of ornithine and phenylacetate.

Where three medicaments are administered, ornithine, phenylacetate andphenylbutyrate are administered at separate times. Ornithine may beadministered first, second or third. Where ornithine is administeredfirst, phenylacetate or phenylbutyrate may be administered second,followed by administration of phenylbutyrate or phenylacetate. Whereornithine is administered second, phenylacetate or phenylbutyrate areadministered first, and phenylbutyrate or phenylacetate are administeredthird. Where ornithine is administered third, phenylacetate orphenylbutyrate are administered first, and phenylbutyrate orphenylacetate are administered second.

The second medicament may be administered up to 5 hours, such as up to 2hours or up to 1 hour, following administration of the first medicament.The second medicament can thus be administered from 15 minutes to 5hours, for example from 30 minutes to 4 hours or from 1 hour to 3 hours,following administration of the first medicament.

The third medicament may be administered up to 5 hours, such as up to 2hours or up to 1 hour, following administration of the secondmedicament. The third medicament can thus be administered from 15minutes to 5 hours, for example from 30 minutes to 4 hours or from 1hour to 3 hours, following administration of the second medicament.

The medicaments of the invention may be administered at the same site orat different sites. The medicaments of the invention may be administeredvia the same route or by different routes. A medicament of the inventionmay be administered by any suitable route. Preferably it is administeredby oral, intravenous, intragastric, intraperitoneal or intravasularroutes. For example, when ornithine and at least one of phenylacetateand phenylbutyrate are administered separately, they may all beadministered orally or they may all be administered intravenously orornithine may be administered orally and the phenylacetate and/orphenylbutyrate may be administered intravenously, or the phenylacetateand/or phenylbutyrate may be administered orally and ornithine may beadministered intravenously.

Therapeutically effective amounts of ornithine, the phenylacetate and/orphenylbutyrate and the optional isoleucine are administered to thesubject. The doses of the ornithine, the phenylacetate and/orphenylbutyrate and the isoleucine can be determined according to variousparameters such as the age, weight and condition of the subject to betreated; the type and severity of the liver disease; the route ofadministration; and the required regimen.

A typical dose of ornithine, of phenylacetate or phenylbutyrate, or ofisoleucine is from 0.02 to 1.25, for example from 0.1 to 0.5, g per kgof body weight, depending on such parameters. Consequently, a dosage ofornithine, of phenylacetate or phenylbutyrate, or of isoleucine may befrom 1 g to 50 g such as from 5 g to 30 g. The dosage of ornithine maybe 10 to 30 g. The dose of isoleucine may be 5 to 15 g. The ornithineand phenylacetate/phenylbutyrate may be administered in a weight ratiofrom 10:1 to 1:10 such as from 5:1 to 1:5 or from 2:1 to 1:2 or about1:1. A physician will be able to determine the required dosage ofornithine and of phenylacetate or phenylbutyrate and of the optionalisoleucine for any particular subject.

A single dose of omithine and a single dose of phenylacetate and/orphenylbutyrate may be administered. Optionally, a single dose ofisoleucine may also be administered. Alternatively multiple doses, forexample two, three, four or five doses, of omithine and/or of thephenylacetate and/or phenylbutyrate and/or of the optional isoleucinemay be administered. Such multiple doses may be administered over aperiod of one month or two weeks or one week. In another embodiment, asingle dose or multiple doses such as two, three, four or five doses ofornithine and/or of phenylacetate and/or phenylbutyrate may beadministered daily.

Other amino acids may be administered to a subject as noted above. Theor each such other amino acid may be administered in the same medicamentas the omithine and/or the phenylacetate and/or phenylbutyrate, or maybe administered separately. When administered separately, the or eachother amino acid may be given simultaneously with, or at a differenttime such as up to 5 hours, up to 2 hours or up to 1 hour before orafter, the administration of omithine and/or phenylacetate and/orphenylbutyrate. The or each other amino acid is typically administeredorally or intravenously.

A therapeutically effective amount of the or each other amino acid isadministered to the subject. The dose will be dependent upon variousparameters such as those noted above for ornithine, phenylacetate andphenylbutyrate. A typical dose of the or each other amino acid is from0.02 to 1.25, for example from 0.1 to 0.5, g per kg of bodyweight. Adosage of the or each other amino acid may therefore be from 1 g to 50 gsuch as 5 g to 30 g.

A single dose of the or each other amino acid may be administered.Alternatively, multiple doses, for example two, three, four or fivedoses may be administered. Such multiple doses may be administered overa period of one month or two weeks or one week. In another embodiment, asingle dose or multiple doses such as two, three, four or five doses maybe administered daily.

The following Examples illustrate the invention.

Example 1 Neutrophil Function is Altered in Patients with Cirrhosis andWorsens with Increasing Severity of Liver Disease Methods forMeasurement of Neutrophil Phagocytosis and Oxidative Burst Phagotest:

Heparinised whole blood was incubated with opsonised FITC-labelled Ecoil and CD16. The cells were then analysed by flow cytometry (FACScanBecton Dickinson), gated through forward and side scatter andsubsequently assessed on the basis of R-phycoerythrin (PE) [Immunotech,Marseille, France] flurochrome expression to identify CD16 positivecells. The gated population was then assessed for the presence ofFITC-labelled bacteria.

Phagoburst:

Heparinised whole blood was incubated with opsonised E coli suspensionto stimulate oxidative burst. A substrate solution was added todetermine the conversion of dihydrorhodamine (DHR) 123 to the flurogeniccompound Rhodamine (R) 123. The reaction was stopped and fixed beforeincubation with CD16 antibody for positive neutrophil identification.Analysis was then undertaken by flow cytometry.

Neutrophil Chemotaxis:

Neutrophil chemotaxis was measured using a modified Boyden chambermethod using interleukin-8 as chemo-attractant to stimulatechemokinesis.

Patients and Methods

We studied 30 patients with cirrhosis (Alcoholic cirrhosis; mean age53.2 (SEM 4.6) and 20 healthy volunteers. Patients with cirrhosis wereclassified as those with superimposed alcoholic hepatitis (AH+) andthose with decompensated or compensated livers. Phagotest was used todetermine the phagocytic capacity and Phagoburst was used to determinewhether the cells were able to generate oxidative burst when exposed toE coli.

Results

We observed that neutrophils from cirrhotic patients had a significantlyreduced ability to phagocytose bacteria. We also found that patientswith cirrhosis had a reduced capacity to respond to stimulation of theneutrophils by E coli in terms of increasing the rate of generation ofoxidative burst (FIG. 1). This reduction in capacity correlated with theseverity of liver disease indicating that the more advanced the stage ofliver disease, the less the ability to respond to and cope withinfection.

Example 2 Ammonia Reduces Phagocytic Capacity in Neutrophils Methods forMeasurement of Neutrophil Phagocytosis and Oxidative Burst

As in Example 1.

Patients and Methods

Blood was collected from healthy volunteers (n=15) and incubated for 1hour with increasing concentrations of ammonia. The ability of theneutrophils to phagocytose bacteria was measured using the Phagotest andNeutrophil chemotaxis assays. 10 ng/ml IL-8 was used in the Neutrophilchemotaxis assay.

Results

With incubation of increasing concentrations of ammonia, there was asignificant reduction in neutrophil phagocytosis (FIG. 2) and also inneutrophil chemotaxis (FIG. 3).

Example 3 The Effect of Ammonia on Neutrophil Phagocytosis can beReversed by Interventions Methods for Measurement of NeutrophilPhagocytosis and Oxidative Burst

As in Example 1.

Patients and Methods

Blood was collected from healthy volunteers (n=15) and incubated for 1hour with ammonia and selected amino acids. The ability of theneutrophils to phagocytose bacteria was measured using the Phagotestassay.

Results

We observed that the ammonia-induced reduction in neutrophilphagocytosis could be partially reversed by ornithine and glutamine(FIG. 4). However, neutrophil phagocytosis was made worse byco-incubation of ammonia with aspartate, but remained unchanged withL-omithine L-aspartate.

Example 4 A Simulated Gastrointestinal Bleed Reduces NeutrophilChemotaxis Which can be Partially Reversed by Administration ofIsoleucine Methods

Ten overnight fasted, metabolically stable patients with biopsy provencirrhosis of the liver [9 males and 1 female; mean 49.6 years (SEM 9.1);mean Child-Pugh score of 7.8 (SEM 1.2)] were studied prior to and twohours after an oral administration of 75 grams of an amino acid mixturethat mimics the hemoglobin molecule (Nutricia, Cuijk, Netherlands). Inseven other patients [4 male and 3 female; mean 51.4 years (SEM 6.7);mean Child-Pugh score of 8.1 (SEM 1.4)], following administration of theamino acid mixture, isoleucine was administered intravenously over a 2hour period (iso-osmotic solution containing 40 mg/l of isoleucine at arate of 100 ml/hr). Neutrophil chemotaxis (see Example 1 for method) andplasma ammonia were measured in peripheral venous blood samples.

Results

Neutrophil chemotaxis was significantly lower in these cirrhoticpatients compared with age-matched controls (53.3 SEM 4.6) and wassignificantly reduced after simulated bleeding from 31 (±4.2) to 8(±5.4) cells/high power field (p<0.0001) (FIG. 5). Plasma concentrationof ammonia increased significantly from 75.1 (±4.2) to 124 (±8.5)(p<0.001). The change in the concentration of ammonia correlated withthe change in neutrophil chemotaxis (r=0.65 and p<0.05). The reductionin neutrophil chemotaxis observed with the simulated bleed was abrogatedin the group of patients treated with isoleucine 25.4 (±6.0) cells/highpower field.

Example 5 A Simulated Bleed Reduces Protein Synthesis and StimulatesIsoleucine Oxidation Inappropriately Methods

Five overnight fasted patients with cirrhosis of the liver wererecruited. A blood sample was collected and expired air was sampledbefore the start of the infusion of the stable isotopes for themeasurement of background isotope enrichment. Then the patients receiveda primed continuous intravenous infusion of [1-¹³C]-isoleucine (1 mg/kgbw/h) until the end of the experiment (t=480 min).

Results

FIG. 6 shows average whole body rate of appearance of isoleucine (Wb Ra)and isoleucine oxidation during the last hour of saline (black bars) andamino acid (grey bars) infusion (values in mean±SEM; # representsp<0.05). An upper GI bleed in patients with cirrhosis resulted in areduction in isoleucine and markedly decreased whole body proteinsynthesis. The fraction of isoleucine flux used for oxidation did notchange after the simulated bleed despite the marked reduction inisoleucine concentration, pointing to occurrence of BCAA antagonism.

Example 6 Administration of Isoleucine During a Simulated Bleed EnhancesProtein Synthesis but does not Reduce Ammonia Concentration Methods

Sixteen metabolically stable patients with biopsy-proven cirrhosis ofthe liver were studied. Patients were randomized either tosupplementation with isoleucine (40 mg/L solution; 50 ml/hr) or placeboduring a simulated bleed over a 4-hour period. Protein synthesis(measured using primed continuous infusion ofL-[ring-²H₅]phenylalanine), L-[ring-²H₄]tyrosine andL-[ring-²H₂]tyrosine) and ammonia.

Results

The results showed that infusion of isoleucine during a simulated bleedin patients with cirrhosis of the liver restores impaired proteinsynthesis of liver and muscle leading to a net anabolic state in theseorgans (Table 1). Ammonia concentration increased significantly in bothgroups but was not significantly different between those administeredwith isoleucine or placebo (FIG. 7).

Example 7 Aspartate Accumulation Following Infusion of L-OrnithineL-Aspartate in Patients with Advanced Cirrhosis Methods

5 patients with advanced cirrhosis who were awaiting livertransplantation (age: 59; 3 male, Child Class C disease, severe ascites,creatinine 102 umol/L) were undergoing treatment with 40 g/day ofL-ornithine L-aspartate.

Results

Over a 3 day period there was a significant and progressive increase inthe aspartate concentration increasing to 5 times the basal value (Table2).

TABLE 2 PRE Day 1 Day 2 Day 3 ASPARTATE 72 (11.8) 178 (23.2) 289 (27.1)354 (31.1) (μmol/L))

TABLE 1 Protein kinetics determined using the Phe model at t = 0 hoursand at study end Time Protein synthesis P Protein breakdown P NetBalance P Liver SB-saline 0 415 ± 120 263 ± 50 152 ± 76  End 274 ± 2500.445 108 ± 162 0.366 166 ± 231 0.836 SB-isoleucine 0 218 ± 37 109 ± 2598 ± 33 End 839 ± 221 0.038 157 ± 204 0.412 682 ± 165 0.010 LegSB-saline 0 117 ± 52 137 ± 51 −20 ± 19  End 372 ± 211 0.189 288 ± 1750.232  87 ± 140 0.694 SB-isoleucine 0 −31 ± 201 196 ± 61 −185 ± 152  End377 ± 135 0.209 159 ± 100 0.535 261 ± 102 0.005 Data are mean ± SEM innmol/kg body cell mass/min. End values represent the mean values of thefinal hour of the amino acid infusion. Protein synthesis data of liverand kidney are corrected for hydroxylation (see methods). Statistics: pvalues for Mann-Whitney U test for differences within groups; nosignificant differences were found between groups

Example 8 Administration with LOLA Reduces Ammonia Concentration butAllows Ammonia to Regenerate Patients and Methods

Eight patients with cirrhosis (age 56 (5.6), 5M, ALD-6; Grade 2 HE: 4;Grade 3-4 HE: 4) were treated with an infusion of LOLA (40 g over 8hours). Blood was sampled for the measurement of ammonia and glutamine.

Results

The results showed that administration of LOLA resulted in a significantreduction in ammonia concentration with a concomitant rise in glutamineconcentration (FIG. 8). This reduction in ammonia had beneficial effectsupon the severity of HE. However, when LOLA was stopped, there was arebound increase in the circulating ammonia levels, resulting inrecurrence of HE in 3 of the 6 patients that had improved.

Example 9 Active Removal of Glutamine Prevents the Secondary Rise inAmmonia Concentration Patients and Methods

3 patients (age 45 (4.1) 2M, ALD, all HE grade 3, FIRS all 3) that wereundergoing heamofiltration (CVVH) were treated with an infusion of LOLA(40 g over 8 hours). Blood was sampled for the measurement of ammoniaand glutamine.

Results

The results showed that LOLA resulted in a reduction in ammoniaconcentration but the addition of dialysis prevented the concomitantincrease in glutamine concentration (FIG. 9). Therefore, we believethere was a sustained reduction in ammonia concentration.

Example 10 Phenylacetate Binds Glutamine to Make an Excretable Compoundand Prevents the Secondary Rise in Ammonia Patients and Methods

6 patients with acute liver failure (5 non-A non-B Hepatitis) and severeencephalopathy (Grade 3-4) were treated with LOLA and phenylacetate (40g/day over 8 hours).

Results

There was no significant increase in glutamine concentration and ammonialevels were reduced with the combined treatment (FIG. 10). No reboundincrease in ammonia was observed.

Example 11 The Effect of Ornithine and Phenylbutyrate in Human Patientswith Hepatic Encephalopathy Patients

1. Groups-3 patients per group. Total 12.2. Inclusion criteria

adult patients aged 18-80 years, —liver cirrhosis documented byhistology or clinical criteria

HE type C, —ammonia concentration of >80 umol/L, informed consent/assent

3. Exclusion criteria

other concomitant neurological disorder, —use of another specificammonia lowering drug, —respiratory failure requiring mechanicalventilation and sedation, —uncontrolled gastrointestinal bleeding,—hypotension requiring inotropes, overt renal failure (creatinine >2mg/dl), hemodialysis, —extracorporeal liver support, knownhypersensitivity to any of the study drugs, —pregnancy.

Assessment of Mental State

Grading of hepatic encephalopathy (West Haven Criteria)

Grade 0 normal mental state (minimal HE) (one or more quantifiableabnormalities on psychometric testing) Grade 1 trivial lack of awarenesseuphoria or anxiety shortened attention span impaired performance ofaddition Grade 2 lethargy or apathy minimal disorientation for time orplace subtle personality change inappropriate behaviour impairedperformance of subtraction Grade 3 somnolence to semi-stupor, butresponsive to verbal stimuli confusion gross disorientation Grade 4 coma(unresponsive to verbal or noxious stimuli)

Methods

In an open labelled study, we included 8 patients with cirrhosis andhyperammonemia. They were matched for the severity of liver disease (seeTable 3). They were treated with one of the following regimes for a 3day period and observations were made for 5 days. The study groups were:

(i) Placebo: 5% Dextrose over 4 hours;

(ii) Ornithine alone: 20 g in 500 ml, 5% dextrose between 0800 and 1200;

(iii) Phenylbutyrate: 10 g twice daily, orally (0800 and 1600); and

(iv) Ornithine+Phenylbutyrate: 20 g in 500 ml, 5% dextrose between 0800and 1200+10 g twice daily, orally (0800 and 1600).

Patients were fasted overnight between 0000 midnight and 0800 am. Theywere fed intragastrically with a diet of 25 KCal/Kg that included 1 g/Kgprotein diet starting at 0800 and finishing at midnight. Blood wassampled at 0730 am and then at 1800 hr for the measurement of ammoniaand glutamine. Patients were monitored closely for side effects. Thedrug was tolerated well in each of the groups and no adverse events wereobserved.

TABLE 3 Patient Demographics Ornithine Phenylbutyrate Placebo alonealone OP Age P1: 47 P3: 46 P5: 56 P7: 52 P2: 57 P4: 40 P6: 48 P8: 52 SexP1: M P3: F P5: F P7: M P2: M P4: F P6: M P8: F Aetiology of P1: HCV P3:HBV P5: NASH P7: HBV Liver Disease P2: HBV P4: NASH P6: HBV P8: HBVSeverity of P1: 9 P3: 13 P5: 14 P7: 14 Liver Disease P2: 12 P4: 13 P6:13 P8: 12 (Pugh Score) Precipitating P1: Infection P3: SBP P5: SBP P7:SBP Factor P2: Infection P4: Infection P6: ?infection P8: InfectionSeverity of HE P1: 2 P3: 3 P5: 3 P7: 3 (West-Haven P2: 3 P4: 3 P6: 3 P8:3 criteria) Severity of HE P1: 9 P3: 8 P5: 9 P7: 9 (Glasgow coma P2: 8P4: 8 P6: 10 P8: 9 score) Other organ P1: none P3: pre-renal, P5: noneP7: none failure P2: hypotension hypotension P6: pre-renal P8: none P4:hypotension Dead/Alive P1: A P3: D P5: A P7: A P2: A P4: A P6: A P8: AComplications P1: infection, SBP P3: HRS P5: sepsis, ICU P7: none P2:infection, P4: rec. P6: recurrent P8: bleed, day variceal bleedinfection SBP 14 SBP: spontaneous bacterial peritonitis, Non alcoholicsteatohepatitis, ICU: Intensive care support needed, HRS: hepatorenalsyndrome

Results

FIG. 11 shows that the mean ammonia levels remained largely unchangedover the period of treatment in the placebo group. In the L-Ornithineand the Phenylbutyrate group, the ammonia concentration increased frombaseline values. In the group treated with both L-omithine andPhenylbutyrate, there was a substantial reduction of ammonia. Thepostprandial increase in ammonia was reduced in the OP treated animalsin addition to the reduction in ammonia concentrations. Both patients inthe OP group had improved their encephalopathy score by 2 grades by day3, which was not observed in any of the other 6 patients.

FIG. 12 shows that the mean glutamine levels remained largely unchangedover the period of treatment in the OP group despite a reduction inammonia. There was a reduction in glutamine in the Phenylbutyrate group,which may well be deleterious. In the L-Ornithine and placebo groupsthere was an increase in Glutamine concentrations which was markedlyaccentuated in the postprandial state.

FIG. 13 shows the changes in mental state in the groups treated withPlacebo, O, P and OP.

Example 12 The Effect of Ornithine, Phenylbutyrate and Isoleucine inHuman Patients with Hepatic Encephalopathy Patients

1. Groups—2 patients per group. Total 62. Inclusion criteria

Adult patients aged 18-80 years, liver cirrhosis documented by histologyor clinical criteria, Child B or C, recent Gastrointestinal bleed fromvarices (<6 hours after presentation), informed consent/assent.

3. Exclusion criteria

other concomitant neurological disorder, use of another specific ammonialowering drug, respiratory failure requiring mechanical ventilation andsedation, uncontrolled gastrointestinal bleeding, hypotension requiringinotropes, overt renal failure (creatinine >2 mg/dl), hemodialysis,extracorporeal liver support, known hypersensitivity to any of the studydrugs, pregnancy/lactation.

Methods

In an open labelled study, we included 6 patients with cirrhosis and whowere admitted for management of variceal bleeding. They were matched forthe severity of liver disease (see Table 4). They were treated with oneof the following regimes for a 3 day period and observations were madefor 5 days. The study groups were:

i. Placebo: 5% Dextrose over 4 hours (250 ml)

ii. Isoleucine alone: 10 gm IV in 250 ml 5% Dextrose over 2 hours in twodivided doses.

iii. Isoleucine+Ornithine+Phenylbutyrate: Isoleucine: 10 gm IV in 250 ml5% Dextrose over 2 hours in two divided doses; Ornithine: 20 g in 250ml, 5% Dextrose (t=0; 24, 48 hr); Phenylbutyrate: 10 g twice daily,orally (t=0, 12, 24, 36, 48 hr).

Patients were fasted overnight between 0000 midnight and 0800 am. Theywere fed intragastrically with a diet of 25 KCal/Kg that included 1 g/Kgprotein diet starting at 0800 and finishing at midnight. Blood wassampled at 0730 am and then at 1800 hr for the measurement of ammoniaand glutamine. Patients were monitored closely for side effects. Thedrug was tolerated well in each of the groups and no adverse events wereobserved. Because the patients received sedation for their initialendoscopy, the mental state assessment was impossible to interpret. Onepatient each in the Placebo and the Isoelucine groups died frommultiorgan failure in the hospital. The rest of the patients survived.

TABLE 4 Isoleucine Placebo alone OIP Age P1: 43 P3: 57 P5: 43 P2: 62 P4:42 P6: 45 Sex P1: M P3: F P5: M P2: M P4: M P6: M Aetiology of Liver P1:ALD P3: HBV P5: HBV Disease P2: HCV P4: ALD P6: NASH Severity of LiverP1: 13 P3: 13 P5: 14 Disease (Pugh P2: 14 P4: 11 P6: 10 Score) Severityof HE P1: 2 P3: 2 P5: 2 (West-Haven P2: 3 P4: 1 P6: 2 criteria)Estimated Blood P1: 9 P3: 7 P5: 7 Loss (u) P2: 10 P4: 8 P6: 10Dead/Alive P1: D P3: A P5: A P2: A P4: D P6: A Complications P1:infection, P3: HRS P5: chest rebleed P4: rec. infection P2: severeinfection P6: none encephalopathy SBP: spontaneous bacterialperitonitis, Non alcoholic steatohepatitis, ICU: Intensive care supportneeded, HRS: hepatorenal syndrome

Results

FIG. 14 shows that no significant changes in ammonia concentrations inthe placebo and the Isoleucine groups. In the group treated with OIP,there was a substantial reduction in ammonia concentration.

FIG. 15 shows that the glutamine levels are not significantly altered byadministration of either Isoleucine, Placebo or OIP. Only in the OIPgroup the ammonia was reduced substantially.

FIG. 16 shows an alternative by which OIP may act is through a reductionin the ammoniagenic amino acid, Glycine. Substantial reduction inGlycine is observed only in the OIP group.

FIG. 17 shows the isoleucine levels are very low to start with in eachof the groups but increases to twice normal values in the Isoleucinetreated groups. The concentration in the Placebo group remains low andunchanged.

FIG. 18 shows the changes in the Ornithine levels in the patients overthe course of treatment showing marked sustained increase in theconcentrations of Ornithine which are significantly reduced to basalvalues on stopping the drug indicating uptake in the different tissues.

Example 13 The effect of ornithine and phenylbutyrate in the bile ductligated rat Methods

Induction of Cirrhosis by Bile Duct Ligation (BDL)

Male Sprague-Dawley rats (200-250 g) were used for this procedure.Following anaesthetisation, a mid-line laparotamy was performed, thebile duct was exposed, triply ligated with 4.0 silk suture, and severedbetween the second and third ligature. The wound was closed in layerswith absorbable suture, and the animal allowed to recover in a quietroom before being returned to the animal storage facility. Animals werekept at a constant temperature (20° C.) in a 12 hour light/dark cyclewith access to water and standard rodent chow ad libitum.

After five weeks post BDL (or sham procedure) the animals were switchedfrom rodent chow to a complete liquid diet (Liquidiet, Bio-Serv,Frenchtown N.J., USA) to which was added an amino acid mixture mimickingthe composition of haemoglobin (2.8 g/Kg/day, Nutricia Cuijk, TheNetherlands, Product No. 24143). At six weeks, under anaesthesia a rightcarotid arterial catheter was inserted and used to collect repeatedblood samples. Following this procedure a baseline sample was collectedprior to administration of the study formulations by IP injection. Thestudy groups were: BDL control+Saline (n=5), BDL+ornithine (0.22 g/Kg,n=6) in saline IP, BDL+phenylbutyrate (0.3 g/Kg, n=7) in saline IP,BDL+OP (0.22 g/Kg/0.3 g/Kg, n=7) in saline IP.

Blood samples were collected into pre-cooled heparinsed tubes and storedon ice prior to processing. Plasma was collected followingcentrifugation (3,000 rpm, 10 mins) and stored at −80° C. prior toanalyses.

Ammonia, glucose, lactate and urea were measured using a COBAS Mira Saccording to manufacturers instructions. Amino acids were quantified byHPLC with fluorescence detection.

Results

In the cirrhotic bile duct ligated rat model there is a substantialincrease in the arterial plasma ammonia level (205±11 μmoles/L,mean±SEM) compared with healthy controls (25.6±2 μmoles/L, p<0.001 data,not shown). In this model we found that there was no change in thearterial ammonia levels over three hours in the saline treated placebogroup.

FIG. 19 shows the change in arterial plasma ammonia levels in BDLcirrhotic rats following IP injections of saline (BDL control, n=5),omithine (Orn, 0.22 g/Kg, n=6), phenylbutyrate (PB, 0.3 g/Kg, n=7) andomithine phenylbutyrate (OP, 0.22 g/Kg+0.3 g/Kg, n=7). * signifiesp<0.05 for OP vs Orn at 3 hours (2 way ANOVA).

This figure shows that in the omithine treated animals a slight decreasein ammonia concentration was detected, though this was not found to bedifferent from placebo. In the phenylbutyrate treated group asignificant increase in plasma ammonia was found after 1 hour (p<0.01 vsall other groups), though this difference was found to be smaller at thethree hour time point. This finding fits with the hypothesis thatphenylbutyrate (phenylacetate) is only effective in subjects with raisedglutamine concentrations. In the animals without ornithinesupplementation which can be metabolised to form glutamine the effectsof P alone are undesirable and are potentially harmful. A significantlylower ammonia level was observed in the ornithine plus phenylbutyrate(OP) treated group. In these animals a sustained lowering of ammonia wasmeasured over the three hour duration of the study the levels of whichwere found to be significantly less than those in the omithine onlygroup at the end of the study (p<0.05).

This clearly demonstrates that the combination of OP has greaterefficacy in reducing plasma ammonia than either O or P alone.Furthermore, the increased plasma levels of ammonia may be detrimentalin the P alone treated animals.

In a subset of samples we examined the uptake of ornithine into theblood stream following IP injection of O or OP. FIG. 20 shows thearterial ornithine concentration in the supplemented groups. It can beclearly seen that in both groups the plasma ornithine concentration ismarkedly increased at 1 hour following the IP injection, which issubsequently reduced at 3 hours as this ornithine is metabolised in thebody. No significant difference was found in plasma ornithineconcentration between these groups at any time point.

This finding is important as it demonstrates that the chosen method ofadministration is effective in delivering ornithine in these animals.Furthermore, the rapid uptake and observed decrease in plasma levelsindicate that active metabolism of this amino acid is occurring.

Example 14 The Effect of Ornithine, Phenylbutyrate and Isoleucine in theBile Duct Ligated Rat Methods

Male Sprague-Dawley rats (200-250 g) were used for this procedure. Forthe 48 hrs prior to sacrifice the animals were switched from standardrodent chow to a complete liquid diet (Liquidiet, Bio-Serv, FrenchtownN.J., USA) to which was added an amino acid mixture mimicking thecomposition of haemoglobin (2.8 g/Kg, Nutricia Cuijk, The Netherlands,Product No. 24143). Acute liver failure (ALF) was induced 24 hours priorto sacrifice by IP injection of galactosamine (1 g/Kg, Sigma, Poole UK)in saline (n=5 in each group). Three hours prior to sacrifice animalswere treated with either a formulation of OIP (ornithine 0.22 g/Kg,isoleucine 0.25 g/Kg, phenylbutyrate 0.3 g/Kg, in saline IP) or salinecontrol. At the termination of the experiment arterial blood wascollected into pre-cooled heparinised tubes and stored on ice untilprocessing. Plasma was collected and stored as above. Ammonia wasdetermined as above.

Results

Arterial ammonia levels were found to be significantly reduced in acuteliver failure rats treated with OIP compared with placebo controls (FIG.21). This study was designed to test whether isoleucine in combinationwith ornithine and phenylbutyrate (phenylacetate) would be able toeffectively lower plasma ammonia. It has been previously demonstratedthat isoleucine alone does not effect ammonia levels in human studies,though its efficacy in combination with O and P has not been previouslytested.

FIG. 21 shows arterial plasma ammonia levels in a hyperammonaemic acuteliver failure model for saline placebo (ALF) and OIP treated (ALF+OIP).A significance level of p<0.01 was found between these two groups(T-Test).

This finding supports the hypothesis that isoleucine in combination withornithine and phenylbutyrate is effective in reducing ammonia levels.These are in addition to the beneficial effects of isoleucine previouslydescribed for protein synthesis.

Example 15 The Effect of Ornithine and Phenylbutyrate in theDevascularized Pig Model Methods

Five pigs were randomised into four groups: acute liver failure(ALF)+placebo+placebo (n=2); ALF+Ornithine+placebo;ALF+Phenylbutyrate+placebo; ALF+Ornithine and Phenylbutyrate. Pigs hadcatheters inserted into the femoral artery and vein, portal vein, renalvein and pulmonary artery. The experiment started at time=−1 hr, whenplacebo or treatment infusions were started.

1. Placebo: 5% Dextrose over 3 hours, oral water placebo

2. Ornithine alone: 0.3 g/Kg, 5% dextrose over 3 hours intravasculardrip

3. Phenylbutyrate: 0.3 g/Kg, 5% dextrose over 3 hours intragastric feedOrnithine+Phenylbutyrate: 0.3 g/Kg, 5% dextrose over 3 hoursintravascular drip, 0.3 g/Kg, 5% dextrose over 3 hours intragastricfeed.

ALF was induced by portal vein anastamosis to the inferior vena cava andsubsequent hepatic artery ligation (devascularisation) at time=0 hr;infusions were stopped at t=+2 hr and the experiment was terminated attime=8 hr. Blood and urine samples were collected at time=0, 1, 3, 5, 7and 9 hr for the measurement of regional ammonia and amino acid changes.At the end of the experiment a section of frontal cortex was removed forbrain water measurements.

Results

Following omithine infusion generating intracellular glutamate and theintragastric supply of conjugating phenylacetate results suggestprofound alteration in overall ammonia levels and glutamine utilizationin this catastrophic model of liver failure.

There is a consistent rise in the arterial ammonia concentration withtime from devascularisation in the placebo treated animal (FIG. 22),with some muscle production (FIG. 23) and a large amount of ammoniacoming from the gut (FIG. 24). This animal shows a modest muscleglutamine release (FIG. 25) and appreciable gut glutamine uptake (FIG.26).

In the case of the ornithine alone treated animal, the early ammoniarise is initially blunted, but rises thereafter to be the highest attermination of the experiment (FIG. 22). There is a net uptake ofammonia by the muscle in this animal (FIG. 24), with a comparable amountof glutamine being released from muscle—compared to the placebo treatedanimal (FIG. 25) with an increased gut uptake of glutamine (FIG. 26).

Phenylbutyrate alone also shows an initial blunting of arterial ammonialevels, which quickly rises to levels comparable with omithine alone atexperiment termination (FIG. 22) with little change in muscle ammoniauptake (FIG. 23), but appreciable gut production of ammonia (FIG. 24).Interestingly, there is a net removal of glutamine by muscle withPhenylbutyrate alone treatment (FIG. 25) with little overt effect on gutglutamine uptake, compared to placebo treated animal (FIG. 26).

The combination of ornithine and Phenylbutyrate has the greatest impacton arterial ammonia levels with an impressive reduction in circulatinglevels at the end of the experiment compared to all the other animals(FIG. 22). Ammonia is actively removed from the blood by muscle in thisanimal (FIG. 23) with a greatly reduced gut ammonia production (FIG.24). It is interesting to note that the muscle glutamine release isincreased compared to both the placebo and ornithine alone treatedanimals (FIG. 25). Despite this increased glutamine production in themuscle the gut glutamine uptake is substantially reduced (FIG. 26).

A demonstration of increased circulating levels of ornithine in theornithine treated animals is shown in FIG. 27.

The impact of the devascularisation and treatment interventions onarterial glutamine are shown in FIG. 28. There is an increase in thecirculating level of glutamine in the ornithine treated animal, which isameliorated by the co-administration of phenylacetate. An interestingfinding was the substantial amelioration of the arterial glycine levelsthat was found in the animal treated with both ornithine andphenylbutyrate (FIG. 29).

At the end of the experiment the frontal cortex of the brain was removedand brain water content measured (FIG. 30).

An independent pathologist reported on the cellular anatomy of the brainin these experimental animals. His report is summarized below.

ALF: Microvessels with perivascular oedema with surrounding vesicles.Neuron with necrotic changes surrounded by vesicles.

ALF+O+P: Microvessels with perivascular oedema with surrounding vesicles(less than from ALF without any treatment). Intracellular edema.

Sham: Brain tissue with minimal ultrastructural changes=normal braintissue.

CONCLUSIONS

The inventors have found that simulation of some of the symptoms of anacute attack associated with chronic liver disease, such as increasingthe concentration of ammonia or simulating a gastrointestinal bleed,results in reduction of neutrophil function and this reduction can bepartially reversed by ornithine or isoleucine. Rescue of neutrophilfunction by both ornithine and isoleucine plays an important role in theprevention of sepsis which is a common precipitating factor in theprogression of liver decompensation.

Furthermore, the inventors have found that isoleucine does not affectthe rise in concentration of ammonia following a simulatedgastrointestinal bleed. Therefore, contrary to the hypothesis thatammonia levels will decrease upon administration of isoleucine becauseof stimulation of protein synthesis, ammonia levels are unaffected.Thus, use of isoleucine in combination with ornithine, which is known tolower ammonia levels, is particularly advantageous.

Therefore, administration of ornithine and isoleucine prevent themetabolic consequences of a gastrointestinal bleed. Rising ammonialevels are blunted, the deficiency in isoleucine is corrected andneutrophil function is rescued. The combined use of ornithine andisoleucine therefore provides a new treatment for patients following aprecipitating event to prevent liver decompensation from occurring.

The inventors have also found that L-omithine L-aspartate (LOLA), whichis used to reduce ammonia in patients with hepatic encephalopathy, doesnot reverse the effect of ammonia on neutrophil function Thus, use ofornithine alone is more advantageous than use of LOLA, since ornithinecan both reduce ammonia and rescue neutrophil function. Also, theaspartate component of LOLA accumulates in the body. This accumulationof aspartate may actually by harmful to patients since aspartate worsensthe effect of ammonia on neutrophil function, further reducingneutrophil function. Accordingly, preventing or delaying the onset ofliver decompensation can be achieved using ornithine in combination withisoleucine, preferably in the absence of aspartate.

Furthermore, the inventors have found that treatment of patients withhepatic encephalopathy (HE) with L-ornithine L-aspartate (LOLA) reducesammonia levels and as a consequence, increases glutamine levels.However, glutamine is only a temporary ammonia buffer as it can recycleand regenerate ammonia in the kidney and the small intestine. Therefore,treatment with LOLA alone can lead to a secondary rise in ammonialevels, further contributing to the pathology of hepatic encephalopathy.

Use of phenylacetate or phenylbutyrate in children with urea cycledisorders reduces the abnormally high levels of glutamine. In contrast,patients suffering from HE have normal levels of glutamine unless, asshown in Example 1, they are being treated with LOLA which reduceslevels of ammonia but increases levels of glutamine. Therefore, use ofphenylacetate and/or phenylbutyrate allows for the removal of glutamineto prevent the secondary rise in ammonia levels in patients with HE.

Accordingly, an improved treatment for hepatic encephalopathy can beachieved by administration of ornithine in combination with at least oneof phenylacetate and phenylbutyrate, preferably in the absence ofaspartate.

Our extensive investigations in animal models and also in humans withcirrhosis support the view that the major organ removing ammonia inpatients with cirrhosis is the muscle, converting ammonia to glutamine,a reaction in which glutamate is utilised. In liver failure, the enzymeresponsible for this reaction, glutamine synthetase is induced and theprovision of glutamate would increase ammonia detoxification.

Ornithine, a precursor of glutamate, detoxifies ammonia bytransformation to glutamine. However, our preliminary studies have shownthat this glutamine, recirculates and regenerates ammonia. Our inventionprovides a novel method of not only detoxifying ammonia into glutaminebut also eliminating the excess glutamine that is generated. Thus, OPreduces ammonia concentration in patients with cirrhosis andhyperammonemia significantly more markedly than either alone. The effectis clearly synergistic rather than additive. In addition, postprandialincrease in ammonia is abolished by administration of OP. This may allowfor feeding of patients with decompensated cirrhosis with protein-richdiets without the risk of hyperammonemia. The reduction in ammonia wasassociated with improvement in the mental state. It achieves reductionin ammonia concentration by preventing an increase in glutamine. This isconsistent with the hypothesis that Ornithine is driving glutamineproduction in the muscle (thereby trapping 1 molecule of ammonia) butthis glutamine is excreted (possibly as an adduct of phenylacetate)preventing a rise in systemic glutamine, thereby preventing reboundhyperammonemia.

The established wisdom that phenylacetate reduces ammonia in thehyperammonaemic infant presenting with urea cycle disorders is that theammonia is trapped into glutamine and that the glutamine is shuttled tothe kidneys for excretion as the phenylacetateglutamine adduct. Theseinfants present with high ammonia and, importantly, high glutamine.Conversely the cirrhotic patient presents with high ammonia and normalto low glutamine. The pig model described above does not have a raisedglutamine and the ammonia levels increase dramatically after the liveris isolated.

Treatment with ornithine alone increases blood glutamine whereas ammonialevels are unaffected. Phenylbutyrate alone marginally increasesglutamine and again has insignificant effects on ammonia levels. Indramatic contrast, in this catastrophic model of escalatinghyperammonaemia the combination of both ornithine and phenylbutyrate(OP) brings about an appreciable reduction in the circulating ammoniaand ameliorates the increase in glutamine seen with ornithine alone.Glycine, an ammonia generating amino acid increased in all the animals,however, the rise in this amino acid was substantially blunted only inthe OP treated animal, suggesting additional benefit for this form ofintervention. An established consequence of elevated ammonia is brainswelling as water content of the brain increases. The brain fromornithine alone treated pig shows considerable increase in water contentwhile the ornithine and phenylbutyrate combined reduces brain watercontent. Histologically, there is less apparent injury in themicrostructure of the brain of the ornithine and phenylbutyrate combinedtreatment animal compared to the placebo treated animal.

1. A pharmaceutical composition comprising ornithine and at least one ofphenylacetate or phenylbutyrate.
 2. The pharmaceutical composition ofclaim 1, wherein the ornithine and at least one of phenylacetate orphenylbutyrate are in a weight ratio from 10:1 to 1:10.
 3. Thepharmaceutical composition of claim 1, wherein the ornithine and atleast one of phenylacetate or phenylbutyrate are in a weight ratio from5:1 to 1:5.
 4. The pharmaceutical composition of claim 1, wherein theornithine and at least one of phenylacetate or phenylbutyrate are in aweight ratio from 2:1 to 1:2.
 5. The pharmaceutical composition of claim1, wherein the ornithine and at least one of phenylacetate orphenylbutyrate are in a weight ratio of about 1:1.
 6. The pharmaceuticalcomposition of claim 1, comprising from 1 g to 50 g of ornithine andfrom 1 g to 50 g of phenylacetate or phenylbutyrate.
 7. Thepharmaceutical composition of claim 1, comprising from 5 g to 30 g ofornithine and from 5 g to 30 g of phenylacetate or phenylbutyrate. 8.The pharmaceutical composition of claim 1, comprising a physiologicallyacceptable salt of ornithine and a physiologically acceptable salt of atleast one of phenylacetate or phenylbutyrate.
 9. The pharmaceuticalcomposition of claim 8, comprising ornithine hydrochloride and at leastone of sodium phenylacetate or sodium phenylbutyrate.
 10. Thepharmaceutical composition of claim 1, further comprising apharmaceutically acceptable carrier or diluent.
 11. The pharmaceuticalcomposition of claim 10, comprising sterile water.
 12. Thepharmaceutical composition of claim 1, formulated for oraladministration.
 13. The pharmaceutical composition of claim 1,formulated for intravenous administration.
 14. The pharmaceuticalcomposition of claim 1, which comprises substantially no other aminoacid.
 15. The pharmaceutical composition of claim 1, which comprisessubstantially no aspartate.
 16. The pharmaceutical composition of claim1, which comprises no aspartate.
 17. The pharmaceutical compositionaccording to claim 1, which further comprises isoleucine.
 18. Apharmaceutical composition consisting essentially of ornithine and atleast one of phenylacetate and phenylbutyrate.
 19. The pharmaceuticalcomposition of claim 18, consisting of ornithine, at least one ofphenylacetate and phenylbutyrate, and a pharmaceutically acceptablecarrier.